Compounds and methods for activating the apoptotic arm of the unfolded protein response

ABSTRACT

N-substituted sulfonylphenyl-5-nitrofuranyl-2-carboxamide derived compounds, which selectively activate the apoptotic, but not the adaptive arm, of the Unfolded Protein Response are provides as is their use in the treatment of diseases such as diabetes, Alzheimer&#39;s, Parkinson&#39;s, hemophilia, lysosomal storage diseases and cancer.

INTRODUCTION

This patent application claims the benefit of priority from U.S.Provisional Application Ser. No. 61/662,099, filed Jun. 20, 2012, thecontents of which are incorporated herein by reference in theirentirety.

This invention was made with government support under contract number 1R03 MH089782-01 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

Defective protein processing within the secretory pathway is an integralcomponent of many genetic and environmental diseases. Diverse diseasestates ranging from diabetes, Alzheimer's disease, and Parkinson'sdisease, to hemophilia and lysosomal storage diseases have all beencharacterized by folding defects or impaired transport from theendoplasmic reticulum (ER). It has been shown that deregulation ofprotein synthesis may be a key component in the pathogenesis of cancerand metastasis (Larsson, et al. (2007) Cancer Res. 67:6814-24; Sorrells,et al. (1999) J. Surg. Res. 85:37-42; Sonenberg & Hinnebusch (2009) Cell136:731-45; Nathan, et al. (1997) Oncogene 15:579-84; Pervin, et al.(2009) Cancer Res. 68:4862-74). When misfolded protein accumulates inthe ER lumen, the cell activates the Unfolded Protein Response (UPR) toclear the misfolded proteins and restore homeostatic protein processing.When a stress is prolonged or robust, the UPR employs a genetic pathwaythat results in cell death.

Stress stimuli that activate UPR include hypoxia, disruption of proteinglycosylation (glucose deprivation), depletion of luminal ER calcium, orchanges in ER redox status (Ma & Hendershot (2004) Nat. Rev. Cancer4:966-77; Feldman, et al. (2005) Mol. Cancer Res. 3:597-605). Theseperturbations result in the accumulation of unfolded or mis-foldedproteins in the ER, which is sensed by resident ER membrane proteins.These proteins activate a coordinated cellular response to alleviate theimpact of the stress and enhance cell survival. Responses include anincrease in the level of chaperone proteins to enhance proteinre-folding, degradation of the mis-folded proteins, and translationalarrest to decrease the burden of proteins entering the ER. Thesepathways also regulate cell survival by modulating apoptosis (Ma &Hendershot (2004) supra; Feldman, et al. (2005) supra; Hamanaka, et al.(2009) Oncogene 28:910-20) and autophagy (Rouschop, et al. (2010) J.Clin. Invest. 120:127-41), and can trigger cell death under conditionsof prolonged ER stress.

Three ER membrane proteins have been identified as primary effectors ofthe UPR: protein kinase R (PKR)-like ER kinase (PERK),inositol-requiring gene 1 α/β (IRE1), and activating transcriptionfactor 6 (ATF6) (Ma & Hendershot (2004) supra). Under normal conditionsthese proteins are held in the inactive state by binding to the ERchaperone GRP78 (BiP). Accumulation of unfolded proteins in the ER leadsto release of GRP78 from these sensors resulting in their activation(Ma, et al. (2002) J. Biol. Chem. 277:18728-35). PERK is a type I ERmembrane protein containing a stress-sensing domain facing the ER lumen,a transmembrane segment, and a cytosolic kinase domain (Shi, et al.(1998) Mol. Cell Biol. 18:7499-509; Sood, et al. (2000) Biochem. J.346(Pt 2):281-93). Release of GRP78 from the stress-sensing domain ofPERK results in oligomerization and autophosphorylation at multipleserine, threonine and tyrosine residues (Ma, et al. (2001) Rapid Commun.Mass Spectrom. 15:1693-700; Su, et al. (2008) J. Biol. Chem.283:469-75). The major substrate for PERK is the eukaryotic initiationfactor 2α (eIF2α) at serine-51 (Marciniak, et al. (2006) J. Cell Biol.172:201-9). This site is also phosphorylated by other PERK familymembers (general control non-repressed 2 (GCN2), PKR, and heme-regulatedkinase) in response to different stimuli, and by pharmacologicalinducers of ER stress such as thapsigargin and tunicamycin.Phosphorylation of eIF2α converts it to an inhibitor of eIF2B, whichhinders the assembly of the 40S ribosome translation initiation complexand consequently reduces the rate of translation initiation. Among othereffects, this leads to a loss of cyclin D1 in cells resulting in arrestin the G1 phase of the cell division cycle (Brewer & Diehl (2000) Proc.Natl. Acad. Sci. USA 97:12625-30; Hamanaka, et al. (2005) Mol. Biol.Cell 16:5493-501). Furthermore, translation of certain messages encodingdownstream effectors of eIF2α, ATF4 and CHOP (C/EBP homologous protein;GADD153), which modulate cellular survival pathways, is increased uponER stress.

A second PERK substrate, Nrf2, regulates cellular redox potential,contributes to cell adaptation to ER stress, and promotes survival(Cullinan & Diehl (2004) J. Biol. Chem. 279:20108-17). The normalfunction of PERK is to protect secretory cells from ER stress.Phenotypes of PERK knockout mice include diabetes, due to loss ofpancreatic islet cells, skeletal abnormalities, and growth retardation(Harding, et al. (2001) Mol. Cell 7:1153-63; Zhang, et al. (2006) Cell.Metab. 4:491-7; Iida, et al. (2007) BMC Cell Biol. 8:38). These featuresare similar to those seen in patients with Wolcott-Rallison syndrome,who carry germline mutations in the PERK gene (Delepine, et al. (2000)Nat. Genet. 25:406-9). IRE1 is a transmembrane protein with kinase andendonuclease (RNAse) functions (Feldman, et al. (2005) supra; Koumenis &Wouters (2006) Mol. Cancer Res. 4:423-36). Under ER stress, it undergoesoligomerization and autophosphorylation, which activates theendonuclease to excise an intron from unspliced X-box binding protein 1(XBP1) mRNA. This leads to the synthesis of truncated XBP1, whichactivates transcription of UPR genes.

The third effector of UPR, ATF6, is transported to the golgi upon ERstress, where it is cleaved by proteases to release the cytosolictranscription domain. This domain translocates to the nucleus andactivates transcription of UPR genes (Feldman, et al. (2005) supra;Koumenis & Wouters (2006) supra).

Tumor cells experience episodes of hypoxia and nutrient deprivationduring their growth due to inadequate blood supply and aberrant bloodvessel function (Brown & Wilson (2004) Nat. Rev. Cancer 4:437-47; Blais& Bell (2006) Cell Cycle 5:2874-7). Thus, they are likely to bedependent on active UPR signaling to facilitate their growth. Consistentwith this, mouse fibroblasts derived from PERK^(−/−), XBP1^(−/−), andATF4^(−/−) mice, and fibroblasts expressing mutant eIF2α show reducedclonogenic growth and increased apoptosis under hypoxic conditions invitro and grow at substantially reduced rates when implanted as tumorsin nude mice (Koumenis, et al. (2002) Mol. Cell Biol. 22:7405-16;Romero-Ramirez, et al. (2004) Cancer Res. 64:5943-7; Bi, et al. (2005)EMBO J. 24:3470-81). Human tumor cell lines carrying a dominant-negativePERK that lacks kinase activity also showed increased apoptosis in vitrounder hypoxia and impaired tumor growth in vivo (Di, et al. (2005)supra). In these studies, activation of the UPR was observed in regionswithin the tumor that coincided with hypoxic areas. These areasexhibited higher rates of apoptosis compared to tumors with intact UPRsignaling. Further evidence supporting the role of PERK in promotingtumor growth is the observation that the number, size, and vascularityof insulinomas arising in transgenic mice expressing the SV40-T antigenin the insulin-secreting beta cells, was profoundly reduced inPERK^(−/−) mice compared to wild-type control (Gupta, et al. (2009) PLoSOne 4:e8008).

Activation of the UPR has also been observed in clinical specimens.Human tumors, including those derived from cervical carcinomas andglioblastomas (Bi, et al. (2005) supra), as well as lung cancers(Jorgensen, et al. (2008) BMC Cancer 8:229) and breast cancers (Ameri,et al. (2004) Blood 103:1876-82; Davies, et al. (2008) Int. J. Cancer123:85-8) show elevated levels of proteins involved in UPR compared tonormal tissues.

Loss of endoplasmic reticulum homeostasis and accumulation of misfoldedproteins can contribute to a number of disease states includingcardiovascular and degenerative diseases (Paschen (2004) Curr. Neurovas.Res. 1(2):173-181) such as Alzheimer's disease (Salminen, et al. (2009)J. Neuroinflamm. 6:41; O'Connor, et al. (2008) Neuron 60(6):988-1009),Parkinson disease, Huntington's disease, amyotrophic lateral sclerosis(Kanekura, et al. (2009) ALS Mol. Neurobiol. 39(2):81-89; Nassif, et al.(2010) Antioxid. Redox Signal. 13(12):1955-1989), myocardial infarction,cardiovascular disease, atherosclerosis (McAlpine, et al. (2010) Cardio.Hematolog. Dis. Drug Targets 10(2):151-157), and arrhythmias.

In a prior screen of ˜66,000 compounds, two thiuram compounds,disulfiram and NSC-1771, were identified as non-selective hits thatcould potently induce both the CHOP (apoptotic) and XBP1 (adaptive) armsof the UPR.

NSC-1771 is used commercially as a fungicide and is known to causedyschondoplasia in the offspring of chickens who consume the grain fromcrops treated with this compound. Therefore, NSC-1771 was notfollowed-up beyond hit validation. Disulfiram(bis(diethylthiocarbamoyl)disulfide) is marketed commercially asANTABUSE and is indicated for aversion therapy to treat chronicalcoholism.

However, because these compounds are not selective, there remains a needin the art for selective activators of the PERK/eIF2α/CHOP (apoptotic),but not the IRE1/XBP1 (adaptive) UPR subpathways.

SUMMARY OF THE INVENTION

This invention features N-substitutedsulfonylphenyl-5-nitrofuranyl-2-carboxamide-derived compounds andmethods for using the same to selectively activate the apoptotic arm ofthe Unfolded Protein Response. An activator of the invention has thestructure of Formula I, or a pharmaceutically acceptable salt, ester orprodrug thereof,

wherein

X is C, N or O; and

R¹ and R² are each independently a hydrogen, hydroxyl, halo, or asubstituted or unsubstituted alkyl or alkoxy group; or

R¹ and R² together form a substituted or unsubstituted cycloalkyl group,

with the proviso that when X is O, R¹ and R² are absent.

A pharmaceutical composition containing a compound of Formula I is alsoprovided, as is a method for synthesizing a compound of Formula I by (a)treating a 4-substituted piperidine with an aryl sulfonyl chloride toproduce a sulfonamide; (b) contacting the sulfonamide with a reducingagent to produce an aniline; and (c) treating the aniline with5-nitrofuran acyl chloride under microwave irradiation.

This invention also provides methods for selectively activating theapoptotic, but not the adaptive arm, of the UPR, and treating orlessening the severity of a UPR-related disease in a subject byproviding a compound of Formula I. In some embodiments, the UPR-relateddisease is a pre-cancerous syndrome, Alzheimer's disease, stroke, Type 1diabetes, Parkinson disease, Huntington's disease, amyotrophic lateralsclerosis, myocardial infarction, cardiovascular disease,atherosclerosis, arrhythmias, age-related macular degeneration or alysosomal storage disorder. In other embodiments, the UPR-relateddisease is cancer. In still other embodiments, the method includes theco-administration of at least one anti-neoplastic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that ML291 induces UPR in a panel of oral squamous cellcarcinoma cells. Shown are the results of qRT-PCR analysis of CHOP,GADD34 and XBP1s after 4 hours of ML291 treatment (5 μM).

FIG. 2 shows that Bax/Bak knockout cells are more resistant to ML291than wild-type cells.

FIG. 3 shows that ML291 inhibits proliferation and induces apoptosis.FIG. 3A, Six OSCC cell lines exposed to ML291 for 24 hours. FIG. 3B,Four ovarian cancer cell lines exposed to ML291 for 24 hours.

DETAILED DESCRIPTION OF THE INVENTION

This invention pertains to N-substitutedsulfonylphenyl-5-nitrofuranyl-2-carboxamide-derived compounds and theiruse in selectively activating the apoptotic, but not the adaptive arm,of the UPR. The compounds of this invention are first-in-class, potent(ML291: 762 nM EC₅₀), not generally cytotoxic, activate genes associatedwith the apoptotic arm of the UPR (by qRT-PCR), and demonstrate efficacyin inducing cell death through activation of the apoptotic arm inrelevant cells. The hit compound for the series was identified through ahigh-throughput screen of the NIH Molecular Libraries Small MoleculeRepository (MLSMR) of >350,000 compounds through complementarycell-based reporter assays using stably transfected CHO-K1 cells thatspecifically identify activators of the PERK/eIF2α/CHOP (apoptotic), butnot the IRE1/XBP1 (adaptive) UPR subpathways. Medicinal chemistry wascarried out to optimize the hit and provide analogs thereof. The hitcompound and its analogs find use in characterizing the molecularmechanism of UPR activation through PERK and ATF6, the removal ofaberrant or pathological “stressed” cells, and in the treatment ofdiseases and conditions such as such as diabetes, Alzheimer's,Parkinson's, hemophilia, cancer (e.g., breast, lung, or head and necksquamous cell carcinoma) and lysosomal storage diseases.

Accordingly, this invention is an N-substitutedsulfonylphenyl-5-nitrofuranyl-2-carboxamide-derived compound of FormulaI, or an analog, stereoisomer, tautomer, pharmaceutically acceptablesalt, or prodrug thereof. Compounds of Formula I have the structure:

wherein

X is C, N or O; and

-   -   R¹ and R² are each independently a hydrogen, hydroxyl, halo, or        a substituted or unsubstituted alkyl or alkoxy group; or

R¹ and R² together form a substituted or unsubstituted cycloalkyl group,

with the proviso that when X is O, R¹ and R² are absent.

As is conventional in the art, a hydroxyl group is —OH; and a halo groupis fluorine, chlorine, bromine or iodine.

As used herein, the term “alkyl” employed alone or in combination withanother term includes a straight or branched chain hydrocarbon. If nototherwise defined, alkyl has 1 to 6 carbon atoms. Examples of “C₁₋₆alkyl” are alkyl residues containing 1, 2, 3, 4, 5, or 6 carbon atomsare methyl, ethyl, propyl, butyl, pentyl, or hexyl, the n-isomers of allthese residues, isopropyl, isobutyl, 1-methylbutyl, isopentyl,neopentyl, isohexyl, sec-butyl, tert-butyl or tert-pentyl.

The term “alkyoxy” includes an alkyl, as defined herein, bonded tooxygen. Methyoxy, ethyoxy, n-propyloxy, isopropyloxy, n-butyloxy,isobutyloxy, sec-butyloxy, tert-butyloxy, n-pentyloxy, isopentyloxy,neo-pentyloxy, n-hexyloxy, isohexyloxy, and the like are exemplified as“alkyoxy.”

A “cycloalkyl” group refers to a saturated closed ring structure with5-7 carbon atoms in the ring. Examples of the cycloalkyl groups arecyclopentyl, cyclohexyl, and cycloheptyl.

If not otherwise defined, alkyl, alkoxy, and cycloalkyl may beunsubstituted or mono, di- or tri-substituted independently of oneanother by groups such as, for example, —F, —Cl, —Br, —I, —CF₃, —NO₂,—CN, —NH₂, —COOH, —OH, —OCH₃, —OCF₃, —CONH₂, -alkyl, or -alkoxy group.

In some embodiments of the invention, X is C, R¹ is hydrogen and R² is ahalo group. In other embodiments, X is C, R¹ is hydrogen and R² is afluoro group. In still other embodiments, X is C, R¹ is hydrogen and R²is a substituted alkyl group. In specific embodiments, the compound ofthe invention is Analog 3 or Analog 4.

Exemplary compounds of Formula I are presented in Examples 10 and 11.The compounds disclosed herein can be used as lead compounds to identifyadditional, structurally related compounds, analogs or derivatives whichactivate CHOP. For example, the compounds disclosed herein can bemodified based upon additional SAR analysis to include additionalsubstituents (e.g., O, N, S, OH, CH₃, halo groups, phenyl groups, alkylgroups, etc.), remove substituents (e.g., O, N, S, OH, CH₃, halo groups,phenyl groups, alkyl groups, etc.), or substitute groups (e.g.,substitute one halo group for another) in order to provide analogs withimproved activity, solubility, and/or efficacy. As with the initialscreens, modified compounds or compound analogs or derivatives can bescreened via in vitro methods to determine activity.

The compounds of the invention can be prepared by the method presentedin Scheme 1, wherein a 4-substituted piperidine of Formula II (wherein Xis C and R¹ and R² are as defined above) is treated with an arylsulfonyl chloride to produce a sulfonamide and the sulfonamide issubsequently contacted with a reducing agent to produce an aniline ofFormula III. A compound of Formula I is then prepared by treating theaniline of Formula III with 5-nitrofuran acyl chloride under microwaveirradiation.

Compounds of the invention can be used as is or prepared aspharmaceutically acceptable salts. As used herein, the term“pharmaceutically acceptable salt” refers to those salts of thecompounds of Formula I which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well-known in the art. See, e.g.,Berge, et al. (1977) J. Pharmaceutical Sciences 66:1-19. Salts can beprepared in situ during the final isolation and purification of thecompounds of the invention, or separately by reacting a free base with asuitable organic acid. Examples of pharmaceutically acceptable saltsinclude, but are not limited to, nontoxic acid addition salts formedfrom amino group and an inorganic acids such as hydrochloric acid,hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid orwith organic acids such as acetic acid, maleic acid, tartaric acid,citric acid, succinic acid or malonic acid or by using other methodsused in the art such as ion exchange. Other pharmaceutically acceptablesalts include, but are not limited to, adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecyl sulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, alkyl having from 1 to 6 carbon atoms, sulfonate and arylsulfonate.

The term “pharmaceutically acceptable prodrug” as used herein refers tothose prodrugs of the compounds of Formula I which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof humans and lower animals with undue toxicity, irritation, allergicresponse, and the like, commensurate with a reasonable benefit/riskratio, and effective for their intended use, as well as the zwitterionicforms, where possible, of the compounds of the present invention.“Prodrug,” as used herein refers to a compound which is convertible invivo by metabolic means (e.g., by hydrolysis) to afford any compounddelineated by the formula of the instant invention. Various forms ofprodrugs are known in the art, for example, as discussed in Bundgaard(ed.) Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.) Methodsin Enzymology, vol. 4, Academic Press (1985); Krogsgaard-Larsen, et al.(ed.) Design and Application of Prodrugs, Textbook of Drug Design andDevelopment, Chapter 5, 113-191 (1991); Bundgaard, et al. (1992) J. DrugDeliv. Rev. 8:1-38; Bundgaard (1988) J. Pharmaceut. Sci. 77:285; Higuchi& Stella (eds.) Prodrugs as Novel Drug Delivery Systems, AmericanChemical Society (1975).

To demonstrate activity, candidate compounds, analogs, stereoisomers,tautomers, pharmaceutically acceptable salts, or prodrugs thereof, canbe tested as described in the Examples herein for their ability toactivate the apoptotic arm of the UPR. The activity of the compounds canbe assayed utilizing methods known in the art and/or those methodspresented herein. For example, quantitative real-timereverse-transcription PCR (qRT-PCR) analysis or northern blot analysiscan be carried out to determine whether CHOP transcripts, e.g., GADD34and proapoptotic BH3-only protein BIM, are induced in the presence of acandidate compound, analog, stereoisomer, tautomer, pharmaceuticallyacceptable salt, or prodrug thereof. Compounds demonstrating the abilityto selectively activate CHOP can be tested in known cell or animalmodels of cancer, Alzheimer's disease, diabetes or a lysosomal storagedisease.

For therapeutic and prophylactic applications, one or more compounds ofthe invention can be formulated as a pharmaceutical composition. Apharmaceutical composition contains a therapeutically effective amountof a compound of this invention formulated together with one or morepharmaceutically acceptable carriers. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator. The pharmaceutical compositions of this invention can beadministered to humans and other animals orally, rectally, parenterally,intracisternally, intravaginally, intraperitoneally, topically (as bypowders, ointments, or drops), buccally, intratumorally,intracerebrally, intracerebroventricularly, or intrathecally andincludes an oral or nasal spray. In particular embodiments, thepharmaceutical composition is a formulation suitable for intratumoral,intracerebral, intracerebroventricular, or intrathecal administration.Such formulations include injectable formulations, implantablereservoirs, infusions or oral or nasal sprays.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups andelixirs. In addition to the active compounds, the liquid dosage formsmay contain inert diluents commonly used in the art such as, forexample, water or other solvents, solubilizing agents and emulsifierssuch as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethylacetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyleneglycol, dimethylformamide, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables.

The injectable formulations can be sterilized, for example, byfiltration through a bacterial-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

In order to prolong the effect of a drug, it is often desirable to slowthe absorption of the drug from subcutaneous or intramuscular injection.This may be accomplished by the use of a liquid suspension ofcrystalline or amorphous material with poor water solubility. The rateof absorption of the drug then depends upon its rate of dissolution,which in turn, may depend upon crystal size and crystalline form.Alternatively, delayed absorption of a parenterally administered drugform is accomplished by dissolving or suspending the drug in an oilvehicle. Injectable depot forms are made by forming microencapsulematrices of the drug in biodegradable polymers such aspolylactide-polyglycolide. Depending upon the ratio of drug to polymerand the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissues.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the compounds of thisinvention with suitable non-irritating excipients or carriers such ascocoa butter, polyethylene glycol or a suppository wax which are solidat ambient temperature but liquid at body temperature and therefore meltin the rectum or vaginal cavity and release the active compound.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the activecompound is mixed with at least one inert, pharmaceutically acceptableexcipient or carrier such as sodium citrate or dicalcium phosphateand/or: a) fillers or extenders such as starches, lactose, sucrose,glucose, mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The active compounds can also be in micro-encapsulated form with one ormore excipients as noted above. The solid dosage forms of tablets,dragees, capsules, pills, and granules can be prepared with coatings andshells such as enteric coatings, release controlling coatings and othercoatings well known in the pharmaceutical formulating art. In such soliddosage forms the active compound may be admixed with at least one inertdiluent such as sucrose, lactose or starch. Such dosage forms may alsocomprise, as is normal practice, additional substances other than inertdiluents, e.g., tableting lubricants and other tableting aids such amagnesium stearate and microcrystalline cellulose. In the case ofcapsules, tablets and pills, the dosage forms may also comprisebuffering agents. They may optionally contain opacifying agents and canalso be of a composition that they release the active ingredient(s)only, or preferentially, in a certain part of the intestinal tract,optionally, in a delayed manner. Examples of embedding compositionswhich can be used include polymeric substances and waxes.

Dosage forms for topical or transdermal administration of a compound ofthis invention include ointments, pastes, creams, lotions, gels,powders, solutions, sprays, inhalants or patches. The active componentis admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, eye ointments, powders and solutionsare also contemplated as being within the scope of this invention.

The ointments, pastes, creams and gels may contain, in addition to anactive compound of this invention, excipients such as animal andvegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulosederivatives, polyethylene glycols, silicones, bentonites, silicic acid,talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the compounds of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the compound in the proper medium. Absorptionenhancers can also be used to increase the flux of the compound acrossthe skin. The rate can be controlled by either providing a ratecontrolling membrane or by dispersing the compound in a polymer matrixor gel.

An inhibitory amount or dose of the compounds of the present inventionmay range from about 0.1 mg/kg to about 500 mg/kg, alternatively fromabout 1 to about 50 mg/kg. Inhibitory amounts or doses will also varydepending on route of administration, as well as the possibility ofco-usage with other agents.

According to the methods of treatment of this invention, a subject, suchas a human or lower mammal, is administered an effective amount of acompound of the invention, in such amounts and for such time as isnecessary to achieve the desired result. The term “effective amount” ofa compound of the invention, as used herein, means a sufficient amountof the compound so as to decrease the signs or symptoms of the diseaseor disorder in a subject. The actual amount effective for a particularapplication will depend, inter alia, on the condition being treated. Forexample, when administered in methods to treat Alzheimer's disease, suchcompositions will contain an amount of active ingredient effective toachieve the desired result (e.g., activating CHOP, protecting againstaberrant mitochondrial and neuronal function, improving learning memory,and/or reducing mitochondrial and cerebral Aβ accumulation). Similarly,when administered in methods to treat cancer, e.g., breast,hematopoietic, lung, laryngeal, pharyngeal and head and neck squamouscell carcinoma, such compositions will contain an amount of activeingredient effective to achieve the desired result of activating CHOPand/or reducing tumor size, growth or formation. Determination of atherapeutically effective amount of a compound of the invention is wellwithin the capabilities of those skilled in the art.

It will be understood that the total daily usage of the compounds andcompositions of this invention will be decided by the attendingphysician within the scope of sound medical judgment. The specifictherapeutically effective dose level for any particular subject willdepend upon a variety of factors including the disorder being treatedand the severity of the disorder; the activity of the specific compoundemployed; the specific composition employed; the age, body weight,general health, sex and diet of the subject; the time of administration,route of administration, and rate of excretion of the specific compoundemployed; the duration of the treatment; drugs used in combination orcontemporaneously with the specific compound employed; and like factorswell known in the medical arts.

The total daily dose of the compounds of this invention administered toa human or other animal in single or in divided doses can be in amounts,for example, from 0.01 to 50 mg/kg body weight or more usually from 0.1to 25 mg/kg body weight. Single dose compositions may contain suchamounts or submultiples thereof to make up the daily dose. In general,treatment regimens according to this invention include administration toa subject in need of such treatment from about 10 mg to about 1000 mg ofthe compound(s) of this invention per day in single or multiple doses.

Lower or higher doses than those recited above may be required. Specificdosage and treatment regimens for any particular subject will dependupon a variety of factors, including the activity of the specificcompound employed, the age, body weight, general health status, sex,diet, time of administration, rate of excretion, drug combination, theseverity and course of the disease, condition or symptoms, the subject'sdisposition to the disease, condition or symptoms, and the judgment ofthe treating physician.

Upon improvement of a subject's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained when the symptomshave been alleviated to the desired level. Subjects may, however,require intermittent treatment on a long-term basis upon any recurrenceof disease symptoms.

As is well known in the art, therapeutically effective amounts for usein humans can also be determined from animal models. For example, a dosefor humans can be formulated to achieve a concentration that has beenfound to be effective in animals. The dosage in humans can be adjustedby monitoring activation of CHOP and adjusting the dosage upwards ordownwards. Adjusting the dose to achieve maximal efficacy in humansbased on animal models and other methods as are well-known in the art iswell within the capabilities of the ordinarily skilled artisan.

Having demonstrated that the compounds of this invention can activatethe apoptotic arm of UPR, this invention also includes a method forselectively activating the apoptotic, but not the adaptive arm, of theUPR. The method involves contacting a cell with an effective amount of acompound disclosed herein so that the apoptotic arm, but not theadaptive arm, of the UPR is activated. Activation of the apoptotic armcan be monitored by determining whether there is a measurable change inthe expression of one or more genes associated with the apoptotic arm ofthe UPR (e.g., BIM, GADD34, endoplasmic oxidoreductin 1-alpha (ERO1α),and Tribbles-related protein (TRB3)) or whether there is a measurableincrease in cell death. In certain embodiments, the cell is a cancercell.

In particular embodiments of this invention, a pharmaceuticalcomposition containing a CHOP activator, pharmaceutically acceptablesalt, ester or prodrug thereof, is useful in the treatment of conditionswherein the underlying pathology is attributable to (but not limited to)modulation of the UPR pathway, for example, cancer and more specificallycancers of the head and neck, breast, colon, lung, pancreas and skin.Not wishing to be bound by theory, it is believed that CHOP-specificactivators will overwhelm the adaptive UPR capacity of malignant cells,while healthy cells, with low or no basal UPR activation, would be ableto mount an effective UPR and overcome the chemotherapeutic challengeand directly induce apoptosis. Accordingly, another aspect of theinvention is directed to methods of treating such UPR-related diseasesor conditions.

In some embodiments, the invention relates to a method for treating orlessening the severity of cancers selected from the group of brain(gliomas), glioblastomas, astrocytomas, glioblastoma multiforme,Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease,Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma,medulloblastoma, lung (e.g., small cell lung cancer, non-small cell lungcancer, squamous cell carcinoma, adenocarcinoma, and large cellcarcinoma), head and neck, kidney, liver, melanoma, ovarian, pancreatic(e.g., insulinomas, adenocarcinoma, ductal adenocarcinoma, adenosquamouscarcinoma, acinar cell carcinoma, and glucagonoma), adenocarcinoma,ductal adenocarcinoma, adenosquamous carcinoma, acinar cell carcinoma,glucagonoma, insulinoma, colon, prostate, breast (e.g., inflammatorybreast cancer, ductal carcinoma, and lobular carcinoma), sarcoma,osteosarcoma, giant cell tumor of bone, thyroid, lymphoblastic T cellleukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia,hairy-cell leukemia, acute lymphoblastic leukemia, acute myelogenousleukemia, chronic neutrophilic leukemia, acute lymphoblastic T cellleukemia, plasmacytoma, immunoblastic large cell leukemia, mantle cellleukemia, multiple myeloma, megakaryoblastic leukemia, multiple myeloma,acute megakaryocytic leukemia, promyelocytic leukemia, erythroleukemia,malignant lymphoma, hodgkins lymphoma, non-hodgkins lymphoma,lymphoblastic T cell lymphoma, Burkitt's lymphoma, follicular lymphoma,neuroblastoma, bladder cancer, urothelial cancer, vulval cancer,cervical cancer, endometrial cancer, renal cancer, mesothelioma,esophageal cancer, salivary gland cancer, hepatocellular cancer, gastriccancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, GIST(gastrointestinal stromal tumor) and testicular cancer.

In other embodiments, the invention relates to a method for treating orlessening the severity of pre-cancerous syndromes in a mammal, includinga human, wherein the pre-cancerous syndrome is cervical intraepithelialneoplasia, monoclonal gammapathy of unknown significance (MGUS),myelodysplastic syndrome, aplastic anemia, cervical lesions, skin nevi(pre-melanoma), prostatic intraepithleial (intraductal) neoplasia (PIN),Ductal Carcinoma in situ (DCIS), colon polyps and severe hepatitis orcirrhosis.

In still other embodiments, the invention relates to a method fortreating or lessening the severity of additional UPR-related diseasesincluding Type 1 diabetes, Alzheimer's disease, stroke, Parkinsondisease, Huntington's disease, amyotrophic lateral sclerosis, myocardialinfarction, cardiovascular disease, atherosclerosis, arrhythmias,hemophilia, age-related macular degeneration, and lysosomal storagedisorder.

The methods of treatment of the invention involve administering aneffective amount of a compound according to Formula I or apharmaceutically acceptable salt, ester or prodrug thereof to a subjectin need thereof. As used herein, “subject” refers to a human or otheranimal. Suitably the subject is a human. By the term “treating” andderivatives thereof as used herein, is meant prophylactic andtherapeutic therapy. Prophylactic therapy is appropriate, for example,when a subject is considered at high risk for developing cancer, or whena subject has been exposed to a carcinogen.

The compounds of Formula I or pharmaceutically acceptable salts, estersor prodrugs thereof may be administered by any suitable route ofadministration, including systemic administration. Systemicadministration includes oral administration, and parenteraladministration. Parenteral administration refers to routes ofadministration other than enteral, transdermal, or by inhalation, and istypically by injection or infusion. Parenteral administration includesintravenous, intramuscular, intraperitoneal injection, and subcutaneousinjection or infusion.

Additionally, the compounds of Formula I or pharmaceutically acceptablesalts, esters or prodrugs thereof may be co-administered with at leastone other active agent known to be useful in the treatment of cancer. Bythe term “co-administration,” as used herein, is meant eithersimultaneous administration or any manner of separate sequentialadministration of a CHOP activating compound, as described herein, and afurther active agent or agents, known to be useful in the treatment ofcancer, including chemotherapy and radiation treatment. The term activeagent or agents, as used herein, includes any compound or therapeuticagent known to or that demonstrates advantageous properties whenadministered to a subject in need of treatment for cancer. Preferably,if the administration is not simultaneous, the compounds areadministered in a close time proximity to each other. Furthermore, itdoes not matter if the compounds are administered in the same dosageform, e.g., one compound may be administered by injection and anothercompound may be administered orally.

Typically, any anti-neoplastic agent that has activity against asusceptible tumor may be co-administered in the treatment of cancer inthis invention. Examples of such agents can be found in CancerPrinciples and Practice of Oncology by Devita & Hellman (Ed.), 6^(th)Edition (2001), Lippincott Williams & Wilkins Publishers. A person ofordinary skill in the art would be able to discern which combinations ofagents would be useful based on the particular characteristics of thedrugs and the cancer involved. Typical anti-neoplastic agents useful inthe present invention include, but are not limited to, anti-microtubuleagents such as diterpenoids and vinca alkaloids; platinum coordinationcomplexes; alkylating agents such as nitrogen mustards,oxazaphosphorines, alkylsulfonates, nitrosoureas, and triazenes;antibiotic agents such as anthracyclins, actinomycins and bleomycins;topoisomerase II inhibitors such as epipodophyllotoxins;anti-metabolites such as purine and pyrimidine analogues and anti-folatecompounds; topoisomerase I inhibitors such as camptothecins; hormonesand hormonal analogues; signal transduction pathway inhibitors;non-receptor tyrosine kinase angiogenesis inhibitors; immunotherapeuticagents; proapoptotic agents; cell cycle signaling inhibitors; proteasomeinhibitors; and inhibitors of cancer metabolism.

The invention is described in greater detail by the followingnon-limiting examples.

Example 1 Materials and Methods

Protocol for UPR-CHOP Primary Assay.

The purpose of this assay was to detect activators of thePERK-eIF2α-CHOP (apoptotic) arm of the Unfolded Protein Responsepathway.

Materials.

DPBS and Heat-inactivated Fetal Bovine Serum were from Hyclone; F12nutrient mix HAMs medium, Penicillin/Streptomycin, liquid, L-glutamine(100×), MEM Non-Essential Amino Acids Solution 10 mM (100×), andTrypsin-EDTA 0.25% were from Invitrogen; T225 TC Flasks were from Nunc;40 μm cell strainers were from BD; 1536-well white solid bottom TCplates were from Aurora Biotechnology; Tunicamycin was fromSigma-Aldrich; and STEADY-GLO Luciferase Assay System was from Promega.

Procedure for Expanding and Maintaining Cells.

CHO-CHOP cells were seeded into T225 flasks at 3.75×10⁵ cells. Cellswere passaged twice a week. Confluency was maintained at <75%. After 3days incubation in 5% CO₂, ˜2.5×10⁷ cells were typically observed perT225 flask. Once the cells had reached confluency, old medium wasaspirated and the flasks were washed with 20 mL DPBS per T225 flask.Cells were left in DPBS for about 30 seconds. Subsequently, 6.5 mL 0.05%trypsin solution was added into the flask and the flask was rockedgently so that the solution covered the surface. The cells were allowedto detach by incubating at room temperature for about 4 minutes. Theflask was washed with mL fresh growth media and the cell suspension wascollected in a 50 mL sterile conical tube. The collected cells werecentrifuged at 900 rpm for 5 minutes and re-suspended in 1 mL freshgrowth media. An additional 19 mL of growth media was added and the tubewas mixed gently. The cell suspension was filtered with a cell strainerand the cells were counted. Stock flasks were prepared with 3.00×10⁵cells per T225 flask for 4 days incubation or 3.75×10⁵ cells per T225flask for 3 days incubation.

Procedure for Preparing Cells for the Screening Assay.

As above, cells were grown to confluency, washed, and detached with 5 mLtrypsin solution for 4 minutes at room temperature. The flask was washedwith 25 mL fresh growth media and the cell suspension was collected in a50 mL sterile conical tube. The cells were centrifuged at 500 rpm for 10minutes and re-suspended carefully in 1 mL fresh assay medium. Anadditional 19 mL of assay media was added the cells were mixed gently.The cell suspension was filtered with a cell strainer and the number ofcells was counted. Cell density was adjusted to 1.0×10⁵ cells/mL inmedium.

Plate Map.

Positive control (P) in columns 1 and 2, 10 μg/mL Tunicamycin; Negativecontrol (N) in columns 3 and 4, No Tunicamycin; and Test compound (T) incolumns 5-48, No Tunicamycin.

Assay Procedure.

On day 1, cell suspensions were prepared and 500 cells in 5 μL of assaymedia were plated in the wells of columns 1-48 of a 1536-well assayplate. The plates were centrifuged at 500 rpm for 1 minute andsubsequently incubated at 37° C., 100% relative humidity, 5% CO₂ for16-18 hours.

On day 2, 25 nL of test compounds (2 mM stock concentration) weretransferred into the wells of the assay plate so that the finalconcentration of the test compounds was 10 μM (0.5% DMSO). The reportercells can tolerate up to ˜4% before UPR activation or toxicity isobserved. The plates were centrifuged at 1000 rpm for 1 minute andsubsequently incubated at 37° C., 100% relative humidity, 5% CO₂ for 6hours.

Following the 6-hour incubation the plate was incubated for 10 minutesat room temperature and 3 μL of STEADY-GLO was added to each well. Theplates were centrifuged at 2000 rpm for 2 minutes and subsequentlyincubated at room temperature for 10 minutes. Luminescence was measuredwith a luminometer.

Example 2 Library Screen for PERK/eIF2α/CHOP Activators

A high-throughput screen of the NIH Molecular Libraries Small MoleculeRepository (MLSMR) of >350,000 compounds was carried out usingcomplementary cell-based reporter assays using stably transfected CHO-K1cells that specifically identify activators of the PERK/eIF2α/CHOP(apoptotic), or the IRE1/XBP1 (adaptive) UPR sub-pathways.

The MLPCN library of approximately 330,000 compounds was tested in theUPR-CHOP primary screen utilizing a luciferase-based reporter expressedin a CHO cell line to identify activators of the CHOP apoptotic pathway.During the performance of the screening campaign, 1125 compounds withactivity≧40% at a single concentration of 10 μM were identified. Liquidsamples for 1125 compounds were obtained.

The liquid samples were first confirmed at a single-point concentrationof 10 μM in the NTR1 HCS primary assay. Of the liquid samples, 674compounds were confirmed to have at least 32% activity at a 10 μM assayconcentration. The confirmed compounds were further tested in doseresponse in the UPR-CHOP primary assay to obtain EC₅₀ values. Inaddition, confirmed compounds were also dosed out in theCHO-XBP1-luciferase reporter cell line to identify compounds that wereselective for the CHOP apoptotic over the adaptive XBP1 pathway.

Chemistry and cheminformatics resources were then employed in theselection of both novel and chemically tractable molecules to pursue fora UPR-CHOP selective probe. Structures of interest and analogs thereofwere obtained. In addition, medicinal chemistry efforts were initiatedon a small group of promising scaffolds.

Example 3 Structure-Activity Relationship (SAR) Analysis

The high throughput screening effort and subsequent validation producedtwo chemotypes, which were further explored by SAR analysis for potency,selectivity, the absence of reactive functionality, syntheticaccessibility, physiochemical properties, and hit rate in unrelatedPUBCHEM assays. SAR testing of re-constituted powders encompassed doseresponse testing of compounds in two assays: UPR-CHOP and UPR-XBP1assays. The sulfonamidebenzamide series and the benzothiazole series,represented by SID 104222717 and SID 104222735, respectively, were foundto have low micromolar potency for CHOP while not exhibiting activity onXBP1 (>70-80 μM).

Both chemotypes were developed in parallel, and the UPR CHOP and XPB1assay data were used to drive the SAR effort. Once promising compoundswere identified that met or were close to meeting the probe criteria(CHOP EC₅₀<1 μM, selectivity over XBP1>10-fold), then compounds wereassessed in more elaborate secondary assays.

For the benzothiazole series, it was clear from the synthesized SAR setthat the 2-pyridyl moiety was necessary to retain CHOP activity, as 3-or 4-pyridyl derivatives lost activity (CHOP EC₅₀>80 μM). Substitutionof the phenyl ring of the benzothiazole with electron withdrawinggroup(s) was also necessary for CHOP activity (e.g., dimethylsubstitution afforded an analog devoid of CHOP activity). However, thecollection of ˜20 analogs revealed a flat SAR with many analogspossessing CHOP EC₅₀ values in the 4.7-9.9 μM range. As a result, thechemotype was deprioritized in favor of more promising results beingobserved with the other scaffold of interest.

For the sulfonamidebenzamide series, modifications first focused on theN-morpholine group of the parent scaffold (Table 1).

TABLE 1

UPR CHOP UPR XBP1 Assay Assay Selectivity Potency^(a) Potency^(b)(XBP1/CHOP) Entry R³ EC₅₀ (μM) EC₅₀ (μM) x-fold 1 N-morpholine 2.28³>80⁴ >35.1 2 3,5-dimethyl-N- 1.36³ >80³ >58.8 morpholine 3 N-piperazine26.43³  >80³ >3.0 4 4-pyran 1.19³ >80² >67.2 5 cyclohexyl 0.68³>80³ >117.6 6 phenyl 1.09³ >80³ >73.4 7 N-pyrrolidine 0.74³ >80³ >108.18 N-piperidine 1.75³ >80⁴ >45.7 9 4-hydroxy-N- 2.02³ >80³ >39.6piperidine 10 4-fluoro-N- 0.77³ >80² >103.9 piperidine 11 4-chloro-N-0.74³ >80² >108.1 piperidine 12 4-methyl-N- 0.54³ >80³ >148.1 piperidine13 4,4-dimethyl-N- 0.49³ >80³ >160 piperidine 14 4-tert-butyl-N- 0.68³>80² >117.6 piperidine 15 4-spirocyclo- 0.73³ >80² >109.6 hexyl-N-piperidine Potency is expressed as mean of replicates. ¹n = 2; ²n = 3;³n = 4; ⁴n = 6. ^(a)AID602434. ^(b)AID602416.

A number of symmetrical, 4-substituted piperidines were evaluated asreplacements for the morpholine ring, as these would provide someinsight into the texture of the SAR and were synthetically readilyaccessible. A polar, 4-hydroxyl group was tolerated without muchfluctuation in CHOP or XBP1 potency, as compared to the unsubstitutedpiperidine (entries 8 (SID104222721) and 9 (SID117695373)). Singlehalogen atoms or a methyl group incorporated in the 4-position of thepiperidine ring afforded analogs with good CHOP potency with EC₅₀ valuesin the 0.77-0.53 μM range while leaving XBP1 unperturbed. Increasingsteric bulk and lipophilic character at that 4-position of the scaffoldculminated in analog SID 117695374 (entry 13) with an EC₅₀=0.50 μM, nodiscernable XBP1 liability and with a C log P of 3.6. Larger, branchedsubstituents occupying the same 4-position, which incidentally alsoincreased the lipophilic character of the corresponding compounds,modestly attenuated CHOP activity (entries 14-15).

This analysis further indicated that hit compound (entry 1;SID104222717), possessed an attractive preliminary profile with CHOPEC₅₀=2.02 μM and XBP1 EC₅₀>71 μM, thus providing a selectivity of35-fold. Shielding the oxygen atom of the morpholine ring with methylgroups had a modest, if any, enhancing effect on the CHOP potency (entry2). Replacement of the oxygen atom for an NH-group (entry 3;SID117695371), a modification done to evaluate the impact of shiftingthe hydrogen-donating character, resulted in dramatically reduced CHOPpotency without affecting XBP1. Removing the nitrogen of the morpholinering was tolerated, and stripping the moiety of heteroatoms generated apromising submicromolar boost in CHOP potency, again, without affectingXBP1 (entries 4 and 5 (SID117695375), respectively).

Several of the analogs derived from these changes were evaluated insecondary assays as they met the preliminary probe criteria for UPR-CHOPand UPR-XBP1 activity. Select compounds were assessed in a dose responsemanner for the ability to suppress proliferation in both the CHOPwild-type and CHOP knockout murine embryonic fibroblasts (−/− MEF KOCHOP)(Table 2). Ideally, active compounds would attenuate proliferationin the wild type cells while leaving the knockout cells unaffected, thusindicating that the observed activity was CHOP pathway specific.

TABLE 2 Proliferation MEF Proliferation −/− Entry wt CHOP MEF KO CHOPClogP 1 NT NT 1.32 2 NT NT 2.36 3 NT NT 1.31 4 NT NT 0.36 5 NT NT 2.75 6NT NT 2.65 7 NT NT 2.01 8 7.84 ± 1.20¹ >20³ 2.57 9 NT NT 0.48 10 17.50 ±2.69²  >20³ 2.26 11 4.77 ± 1.05³ >20³ 2.70 12 4.57 ± 1.24² 16.30 ±0.99²  3.09 13 3.02 ± 0.60² 9.17 ± 3.34² 3.61 14 1.36 ± 0.54² 2.05 ±0.29² 4.41 15 NT NT 4.56 Potency is expressed as mean of replicates. ¹n= 2; ²n = 3; ³n = 4. NT = not tested.

For those compounds which were tested, increasing anti-proliferativeeffects correlated with increasing C log P in wild-type cells; however,a similar trend in toxicity was also observed in the knockout cells. The4-chloro-N-piperidine derivative (entry 11) proved to have the bestprofile with a CHOP wt EC₅₀=4.77 μM and no observed effect (EC₅₀>20 μM)on −/− MEF knockout cells.

Nitrofurans are a class of compounds characterized by a nitro functionalgroup (NO₂) attached to a furan heterocycle. The nitro functionality isa 1,3-dipolar group with delocalized charge spread over three atoms.Although UPR activation was observed in xenografts, nitro features indrug scaffolds may reduce absorption and tissue penetration and arylnitro groups are often quickly metabolized in the liver. Therefore,drugs containing this structural element are typically used topically orto treat infections that do not require tissue penetration such ascystitis (urinary tract infection) and diarrheal illnesses. While, aprodrug approach has been used to overcome some of the ADMET liabilities(Chung, et al. (2011) Curr. Pharmaceut. Design 17:3515-3526;Tangallapally, et al. (2007) Curr. Top. Med. Chem. 7:509-526),modification of the furan group was also carried out (Table 3).

TABLE 3

UPR UPR CHOP XBP1 Selectivity Assay Assay (XBP1/ Potency^(a) Potency^(b)CHOP) Entry R³ R⁴ EC₅₀ (μM) EC₅₀ (μM) x-fold 1 morpholine 5-nitro-2-   2.28² >80³ >35.1 furan 2 morpholine 5-nitro-2-   13.5² >80³ 5.9thiophene 3 morpholine 2-thiophene >80² >80³ 1.0 4 morpholine Phenyl>80² >80³ 1.0 5 morpholine 4- >80² >80² 1.0 nitrophenyl 6 4,4- 3- >80²>80¹ 1.0 dimethyl- nitrophenyl N- piperidine 7 N-4,4- 1-methyl-5-  77.52² >80¹ 1.0 dimethyl- nitro-1H-2- N- imidazole piperidine Potencyis expressed as mean of replicates. ¹n = 3; ²n = 4; ³n = 6.^(a)AID602434. ^(b)AID602416.

Replacing the 5-nitro-2-furan with a bioisosteric 5-nitro-2-thiopheneresulted in a 6-fold loss in CHOP potency (entry 2; SID 104222733).Removal of the nitro group from the 2-thiophene group afforded an analogdevoid of any activity. Phenyl derivatives, with or without the nitrofunctionality, were not tolerated (entries 4-6). A nitroimidazole analogwas also found to be inferior to the parent 5-nitro-2-furan moiety(entry 7). Based on these results, an attempt was made to remove orreplace the nitro group while preserving the furan functionality (Table4).

TABLE 4

UPR UPR CHOP XBP1 Selectivity Assay Assay (XBP1/ Potency^(a) Potency^(b)CHOP) Entry R³ R⁵ EC₅₀ (μM) EC₅₀ (μM) x-fold 1 N-morpholine NO₂    2.28²>80³ >35.1 2 N-morpholine H >80² >80³ 1.0 3 N-morpholine Methyl >80²>80³ 1.0 4 N-morpholine Bromine >80² >80³ 1.0 5 4,4-dimethyl- CF₃ >80²>80¹ 1.0 N-piperidine Potency is expressed as mean of replicates. ¹n =3; ²n = 4; ³n = 6. ^(a)AID602434. ^(b)AID602416.

Removal of or exchange of the nitro group for methyl, bromine, ortrifluoromethyl appendages resulted in complete loss of CHOP activity.

The SAR analysis indicated that the nitrofuran moiety was necessary foractivating the CHOP pathway and that changes to the sulfonamideappendage permitted modulation of the lipophilicity and CHOP potencywithout affecting XBP1. Several compounds were identified withsubmicromolar CHOP activity and no discernable XBP1 liability.Subsequent analysis to prove that the observed effects wereCHOP-pathway-specific revealed that some of the active analogs weretoxic to cells due to non-related CHOP pathways and this trended withincreased lipophilicity. The overall profile of the4-chloro-N-piperidine derivative (Table 1, entry 11) was of interest, asit possessed submicromolar CHOP activating potency and excellentselectivity against XBP1 (EC₅₀=0.74 μM, XBP1 EC₅₀>80 μM,selectivity >108.1×). Moreover, it demonstrated desirable CHOP-pathwayselective toxicity with a CHOP wild-type EC₅₀=4.77 μM and no observedeffect (EC₅₀>20 μM) on −/− MEF knockout cells. As such, this compound(ML291) was selected as a suitable probe with which furtherinterrogation of the CHOP pathway could be explored.

Example 4 Cellular Activity of ML291

It was determined whether ML291 and ML291 congeners would induce celldeath (cytotoxicity as measured by ATPLite®, which measures productionof ATP from living cells) in cells where these pathways are operant, butnot activate them in the absence of the pathways. Using mouse embryonicfibroblast (MEF) cell lines engineered to have the wild-type apoptoticpathways intact (MEF with wild-type CHOP) and MEF cells where thispathway has been knocked-out (MEF with CHOP KO), it was demonstratedthat ML291 did not non-specifically kill MEFs; however, ML291 didpotently kill MEFs in the presence of an intact CHOP pathway (Table 5),thereby demonstrating activation of apoptotic pathways. In a separateseries of experiments, it was estimated that the activator potency ofML291 in MEF wild-type CHOP was ˜4.8 μM EC₅₀ (n=4), which comparedfavorably (˜6-fold shift) with its potency in the CHO-K1 cell reporterassays (762 nM EC₅₀).

TABLE 5 Average IC₅₀ (μM) Wild-Type Compound CHOP CHOP KO

7.84 ± 1.20 >20

17.5 ± 2.69 >20

4.77 ± 1.05 >20

4.57 ± 1.24 16.3 ± 0.99

3.02 ± 0.60 9.17 ± 3.34

1.36 ± 0.54 2.05 ± 0.29

Example 5 Profiling Assays

ML291 was evaluated in a detailed in vitro pharmacology screen. Theresults of this analysis are presented in Table 6.

TABLE 6 Aqueous Solubility in pION's buffer 3.6/3.8/4.0 (μg/mL) [μM]^(a) pH 5.0/6.2/7.4 [8.7/9.2/9.7] Aqueous Solubility in 1x PBS, pH 7.43.9 [9.4] (μg/mL) [μM] ^(a) PAMPA Permeability, P_(e) (×10⁻⁶ cm/s) Donor291/329/245 pH: 5.0/6.2/7.4 Acceptor pH: 7.4 Plasma Protein BindingHuman 1 μM/10 μM 99.75/99.63 (% Bound) Mouse 1 μM/10 μM 84.47/84.35Plasma Stability (% Remaining at 3 hours @ 85.58/52.31 37° C.)Human/Mouse Hepatic Microsome Stability (% Remaining 0.49/0.02 after 1hour @ 37° C.) Human/Mouse Toxicity Towards Fa2N-4 Immortalized 11.4Human Hepatocytes LC₅₀ (μM) ^(a)Solubility also expressed in molar units(μM) as indicated in italicized [bracketed values], in addition to moretraditional μg/mL units.

ML291 had poor-to-modest solubility ranging from 8.7-9.7 μM (3.6-4.0μg/mL) in aqueous buffers between a pH range of 5.2-7.4. Solubility washighest at the physiological of pH 7.4. This solubility was about 11-13fold over its EC₅₀ (762 nM), so its apparent potency was not severelylimited by its solubility.

The PAMPA (Parallel Artificial Membrane Permeability Assay) assay wasused as an in vitro model of passive, transcellular permeability. Anartificial membrane immobilized on a filter was placed between a donorand acceptor compartment. At the start of the test, drug was introducedin the donor compartment. Following the permeation period, theconcentration of drug in the donor and acceptor compartments wasmeasured using UV spectroscopy. ML291 exhibited good permeability at pHsof 5.0, 6.2 and 7.4 in the donor compartment, with highest permeabilityat pH 6.2.

Plasma protein binding is a measure of a drug's efficiency to bind tothe proteins within blood plasma. The less bound a drug is, the moreefficiently it can traverse cell membranes or diffuse. Highly plasmaprotein bound drugs are confined to the vascular space, thereby having arelatively low volume of distribution. In contrast, drugs that remainlargely unbound in plasma are generally available for distribution toother organs and tissues. ML291 was highly plasma protein bound to humanplasma proteins (>99%), though it is somewhat less tightly bound tomouse plasma proteins (˜84%).

Plasma stability is a measure of the stability of small molecules andpeptides in plasma and is an important parameter, which strongly caninfluence the in vivo efficacy of a test compound. Drug candidates areexposed in plasma to enzymatic processes (proteinases, esterases), andthey can undergo intramolecular rearrangement or bind irreversibly(covalently) to proteins. ML291 appeared to be moderately stable inhuman plasma (˜84% remaining), but less so in mouse plasma (˜52%remaining).

The microsomal stability assay is commonly used to rank compoundsaccording to their metabolic stability. This assay addresses thepharmacologic question of how long the parent compound will remaincirculating in plasma within the body. ML291 was almost completelymetabolized in both human and mouse liver homogenates within 1 hour.

ML291 was submitted to Ricerca Biosciences LLC (Bothell, Wash.) toevaluate it in radio-ligand binding assays to determine activity againsta panel of 67 GPCRs, ion channels and transporters at 10 μM. ML291 onlyscored as having significant activity (68% inhibition) against thedopamine transporter (DAT), so it did not appear to be generallypromiscuous compound with respect to these receptors.

Example 6 Mechanism of Action Analyses

While ML291 robustly and selectively activated the apoptotic (CHOP) armof UPR in the engineered luciferase reporter CHO-K1 cells, while notactivating reporter under the control of the adaptive arm (XBP1)promoters, direct confirmation of the selective activation of genes andpathways on the apoptotic UPR arm in a biologically relevant cell orcell line was needed. To determine whether or not ML291 couldselectively activate the apoptotic (CHOP) arm of the UPR, relevant oralsquamous cell carcinoma cell lines, UMSCC23, A253 and HN12, were treatedwith ML291 for four hours. The activation/induction of associated genesalong the UPR sub-pathways were measured by quantitative real-timereverse-transcription PCR (qRT-PCR) analysis of cDNA pools generatedfrom whole cell lysates. These transcription profile studies revealed a5-fold selectivity of CHOP transcripts (apoptotic) compared to splicedXBP1 (adaptive). Furthermore, the simultaneous induction of the CHOPdown stream target GADD34, but not the DNA excision and repair geneERCC1 indicated ML291 was activating CHOP via the UPR and not DNAdamage. This analysis further revealed an accumulation of CHOP, GADD34and spliced XBP1 transcripts (FIG. 1). Immunoblot analysis of whole celllysates revealed an accumulation of CHOP, ATF4, GADD34 and thephosphorylated form of eIF2α (p-eIF2α), a hallmark of ER stress,supporting the gene expression assays. Identically treated cellsdemonstrated an accumulation of ubiquitinated proteins indicating thecells were working to resolve a burden of unfolded proteins through theubiquitin-proteasome system. These data definitively show that ML291 canselectively activate CHOP via a mechanism governed by the UPR apoptoticarm. These data, coupled with the observation that ML291-treated MEFcells, with intact wild-type CHOP, undergo apoptosis while ML291-treated−/− MEF cells (CHOP knock-out) show lack of cytotoxicity, indicate thatML291 may selectively activate apoptotic pathways in tumors therebyleading to their selective and efficacious killing.

Example 7 Animal Model of Oral Squamous Cell Carcinoma

Stable oral squamous cell carcinoma (OSCC) cell lines that expressluciferase constructs for XBP1 and human CHOP were generated. The XBP1cell line (UMSCC23-XBP1-luc) was generated with the same XBP1 plasmidused to make the CHO-XBP1 cell line used in the primary assay. The CHOPcell line (UMSCC23-hCHOP-luc) was generated with a novel plasmid createdby cloning ˜3 kb of the human CHOP promoter to a luciferase gene. TheChop promoter driving the expression of luciferase in the CHO cell lineused for screening was murine; the 26 bp intron that is spliced fromXBP1 (by IRE1α) is conserved between murine and human. Singlehigh-expressing clones were identified and used successfully to createxenografts (in SCID mice) that express luciferase when stimulated withtunicamycin or other UPR-inducing compounds. Using this model, theability of ML291 to activate the UPR (i.e., CHOP gene activation and/orXBP1 mRNA splicing) can be observed in vivo in real-time. Additionally,the ability of ML291 to reduce tumor burden or interfere with tumorgrowth can be quantitatively measured over time. In vivo luminescencewas analyzed by IP injection of luciferin and imaging on a CARESTREAMIn-Vivo Xtreme multi-modal optical and X-ray small animal imagingsystem. Bioluminescent images of xenografts made with the stable OSCCcell line A253-CHOP-luc demonstrated increased luminescence 24 hoursafter a 10 mg/kg (IP) dose of ML291. Immunoblots with tumor lysatedemonstrated increased BiP, CHOP and also p-eIF2α. These data indicatethat ML291 induces ER stress and the UPR in vivo.

To confirm the ability of ML291 to activate UPR-luciferase reportersstably transfected into UMSCC23 and A253 cells and reduce tumor burden,additional xenograft studies are performed with additional animals topermit real-time measurement of UPR activation during drug treatment(bioluminescent imaging). The data presented herein indicate that ML291can activate the UPR in xenograft tissue at 10 mg/kg. However,pharmacokinetic studies (IV and IP) suggest that further chemicaloptimization might improve activity. Thus, mice are injected IP in themorning with 10-100 mg/kg ML291. Injection in the morning, i.e., whenthe lights come on in the vivarium and the mice stomachs are most likelyto be full, is a technique known to enhance absorption of nitrofuransinto the bloodstream (Heinrichs (2001) Behavioural Brain Res.125:81-88). Preliminary experiments revealed no differences in weight,physical appearance, disposition or activity between mice that were IPinjected with 10 mg/kg ML291 daily and controls. Upon completion of thisexperiment, control and ML291 treated mice (3 each) undergo completepathological necropsy to identify gross or histological signs of organtoxicity. Once the degree to which ML291 induces the UPR and reducesxenograft burden is analyzed, additional studies are carried out withcombination therapies using standard of care OSCC drugs. Preliminarycombination studies with cisplatin and taxol demonstrated reduced IC₅₀values for ML291 and enhanced toxicity. It is posited thatML291-enforced UPR in the tumor milieu potentiates conventionaltherapies and might enhance the effects of cisplatin, ifosfamide ortaxol.

Example 8 Apoptotic Cell Death in OSCC and Ovarian Cancer

Cells

To determine whether ML291 could reduce the proliferation of malignantcells, a panel of OSCC (UMSCC23, A253, HN6, HN12, HN30, H460/T800) andovarian cancer (RMG-1, TOV112D, A2780 and SKOV3) cell lines, weretreated with increasing doses (0.15-20 μM) of ML291 for 24 hours (FIGS.3A and 3B). ML291 reduced proliferation in eight of the ten cell linesdemonstrating IC₅₀ values 1.6-18.9 μM (Table 7).

TABLE 7 Cell Line Average IC₅₀ (μM) UMSCC23 7.31 ± 1.68* A253 17.2 ±3.64* HN6 >20*    HN12 7.17 ± 0.69* HN30 8.95 ± 1.06* H460/T800 18.9 ±1.83* RMG-1 (ovarian) >20    TOV112D (ovarian) 6.59 A2780 (ovarian) 2.16SKOV3 1.59 *Average of three experiments.

Although primary or non-malignant cells were not examined, the fact thattwo cell lines were resistant (up to 20 μM), and pharmacokinetic studieswith animals injected bi-weekly with 10 mg/kg for four weeks did notdisplay any obvious side effects, indicates that ML291 is not generallycytotoxic.

To identify genes that confer resistance to apoptotic UPR signaling andto further elucidate the mechanisms by which this novel chemical seriesinduces a terminal UPR, a genome-wide RNAi screen in OSCC cells isperformed. This approach employs a pool of ˜200,000 HIV-based virusesthat target the entire human genome with at least 5 unique shRNAsequences per gene. Pooled infected cells are puro-selected and treatedwith 10 μM ML291 (i.e., the IC₅₀ dose) or vehicle for 24 hours. GenomicDNA is collected from half of the cells, the other half is expanded andre-exposed to ML291 or vehicle for another 24 hours and genomic DNA iscollected. Genomic DNA from the ML291-surviving and vehicle-treatedcells is hybridized to an AFFYMETRIX Human Genome U133 Plus 2.0 Array toidentify genes able to interfere with ML291-induced cytotoxicity. It hasbeen reported that double selection results in a 300-fold enrichment intarget effected genes identified through shRNA screening (Bassik, et al.(2009) Nat. Meth. 6:443-445). The shRNA sequences hybridize to the arrayin a fashion that the count of hits for a specific gene corresponds tothe number of cells surviving with that specific shRNA. To confirm theresults of the shRNA screen lentiviruses that express specific shRNA'sor pools of siRNA's in a panel of human cancers are used. Alternatively,or in addition to, targeted genome editing can be used to knockout anygene in the genome with highly active TALEN's of Zinc Finger Nucleases.

To determine if apoptosis might contribute to ML291-reducedproliferation, immunoblot experiments were performed with three OSCCcell lines. Pro-caspase 9 levels were decreased by 8 hours, an affectthat was followed by accumulation of Caspase 3 and cleaved PARP. NOXA,PUMA, BAK and BAX levels increased during the same time course,implicating the pro-apoptotic BH3-only BCL2 family in ML291proliferation inhibition.

qRT-PCR profiling with HN12 cells identified 17 apoptosis-related genesthat were upregulated following ML291 treatment including BAX, BCL10,BIRC2, CARD6, CARD8, CASP9, CD40, CRADD, DAPK1, DFFA, FAS, FASLG, TNF,TNFSF10, CD70, TP53BP2 and TP73.

Furthermore, the human Zinc Finger Nuclease-deleted colorectal cell lineDLD1(BAX^(−/−))(BAK^(−/−)) was significantly more able to withstandML291 exposure than wild-type controls (FIG. 2). These data indicatethat ML291 can induce apoptosis and reduce the proliferation of OSCCcells in culture.

Example 9 Synthesis and Physical Properties of ML291

The IUPAC name of the probe ML291 isN-(4-((4-chloropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide.

The physiochemical data for ML291 is presented in Table 8.

TABLE 8 Molecular Formula C₁₆H₁₆ClN₃O₆S Molecular Weight 413.83 g/molExact Mass 413.04 g/mol CLogP  2.70 Topological Polar Surface Area127.52 Physical State Off-White Solid Melting Point 228-232° C.

Structure Verification and Purity.

¹H and ¹³C NMR spectra were recorded on a Bruker AM 400 spectrometer(operating at 400 and 101 MHz, respectively) or a Bruker AVIIIspectrometer (operating at 500 and 126 MHz, respectively) in CDCl₃ with0.03% TMS as an internal standard or DMSO-d₆. The chemical shifts (δ)reported are given in parts per million (ppm) and the coupling constants(J) are in Hertz (Hz). The spin multiplicities are reported ass=singlet, br. s=broad singlet, d=doublet, t=triplet, q=quartet,dd=doublet of doublet and m=multiplet. Liquid chromatography-massspectrometry (LCMS) analysis was performed on an Agilent 1200 RRLchromatograph with photodiode array UV detection and an Agilent 6224 TOFmass spectrometer. The chromatographic method utilized the followingparameters: a Waters Acquity BEH C-18 2.1×50 mm, 1.7 μm column; UVdetection wavelength=214 nm; flow rate=0.4 ml/min; gradient=5-100%acetonitrile over 3 minutes with a hold of 0.8 minutes at 100%acetonitrile; the aqueous mobile phase contained 0.15% ammoniumhydroxide (v/v). The mass spectrometer utilized the followingparameters: an Agilent multimode source which simultaneously acquiresESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of90:10:0.1 MeOH:Water:Formic Acid, which was introduced to the LC flowprior to the source to assist ionization. Melting points were determinedon a Stanford Research Systems OptiMelt apparatus.

The probe was prepared as depicted in Scheme 2.

4-Chloro-1-((4-Nitrophenyl)Sulfonyl)Piperidine

To a vial was added 4-nitrobenzenesulfonyl chloride (0.32 g, 1.4 mmol),pyridine (0.11 g, 1.4 mmol) and THF (1.5 mL). The reaction was stirredat room temperature while 4-chloropiperidine (0.13 g, 1.0 mmol) wasadded drop-wise over 10 minutes. The reaction was subsequently heated to60° C. for 20 minutes and monitored by thin layer chromatography (TLC).Upon completion, the reaction was cooled to room temperature, dilutedwith EtOAc (10 mL) and washed with saturated aq. NaHCO₃ (10 mL). TheEtOAc layer was separated, dried with MgSO₄, filtered, adsorbed tosilica and purified by silica gel flash column chromatography (15minutes, 0-30% v/v EtOAc/hexanes) to produce pure4-chloro-1-((4-nitrophenyl)sulfonyl)piperidine (0.29 g, 0.96 mmol, 96%yield). ¹H NMR (400 MHz, CDCl₃): δ 8.40 (d, J=9.0 Hz, 2H), 7.96 (d,J=9.0 Hz, 2H), 4.22 (m, 1H), 3.29 (m, 2H), 3.18 (m, 2H), 2.16 (m, 2H),1.97 (m, 2H).

4-((4-Chloropiperidin-1-yl)Sulfonyl)Aniline

To a vial containing 4-chloro-1-((4-nitrophenyl)sulfonyl)piperidine(0.29 g, 0.96 mmol) was added 1:1 MeOH:CH₂Cl₂ (3 mL:3 mL), and thereaction was cooled to 0° C. Raney nickel (0.006 g, 0.096 mmol) wasadded followed by portion-wise addition of sodium borohydride (0.073 g,1.9 mmol). Once addition was complete, the reaction mixture was stirredat 0° C. for 30 minutes and was then diluted with CH₂Cl₂ (10 mL) andfiltered slowly. The CH₂Cl₂ layer was washed with water (10 mL),separated, dried with MgSO₄, filtered, adsorbed to silica and purifiedby silica gel flash column chromatography (15 minutes, 0-5% v/vMeOH/DCM) to produce pure 4-((4-chloropiperidin-1-yl)sulfonyl)aniline(0.23 g, 0.82 mmol, 86% yield). ¹H NMR (400 MHz, CDCl₃): δ 7.66 (d,J=8.7 Hz, 2H), 7.06 (d, J=8.6 Hz, 2H), 7.03 (s, 1H), 5.32 (s, 1H), 4.12(m, 1H), 3.16 (m, 2H), 3.10 (m, 2H), 2.13 (m, 2H), 1.95 (m, 2H).

N-(4-((4-Chloropiperidin-1-yl)Sulfonyl)Phenyl)-5-Nitrofuran-2-Carboxamide(ML291)

To a microwave vial was added4-((4-chloropiperidin-1-yl)sulfonyl)aniline (0.23 g, 0.82 mmol),5-nitro-2-furoyl chloride (0.16 g, 0.90 mmol) and acetonitrile (3 mL).The vial was sealed and heated to 150° C. in the microwave for 20minutes. The reaction then cooled to room temperature and was dilutedwith CH₂Cl₂ (10 mL) and washed with saturated NaHCO₃ (10 mL). The CH₂Cl₂layer was separated, dried with MgSO₄, filtered and adsorbed to silicagel. The crude product was purified by silica gel flash columnchromatography (20 minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pureN-(4-((4-chloropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide,ML291 (0.11 g, 0.26 mmol, 31% yield). ¹H NMR (400 MHz, DMSO-d₆: δ 11.01(s, 1H), 8.03 (d, J=8.8 Hz, 2H), 7.83 (d, J=3.9 Hz, 1H), 7.79 (d, J=8.8Hz, 2H), 7.70 (d, J=3.9 Hz, 1H), 4.27 (m, 1H), 3.17 (m, 2H), 2.87 (m,2H), 2.10 (m, 2H), 1.79 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆): δ 154.9,151.9, 147.3, 142.2, 130.4, 128.6, 120.4, 117.2, 113.4, 56.1, 43.4,33.9. LCMS retention time: 3.147 min. LCMS Purity at 214 nm: 97.5%.HRMS: m/z calcd for C₁₆H₁₇ClN₃O₆S (M+H⁺) 414.0521. found 414.0522.Melting point: 228-232° C.

Solubility.

Solubility was measured in phosphate-buffered saline (PBS) at roomtemperature (23° C.). PBS by definition is 137 mM NaCl, 2.7 mM KCl, 10mM sodium phosphate dibasic, 2 mM potassium phosphate monobasic and a pHof 7.4. Probe ML291 was found to have a solubility measurement of 3.9μg/mL, or 9.4 μM, under these conditions.

Stability.

Stability was measured under two distinct conditions with ML291: at roomtemperature (23° C.) in PBS (no antioxidants or other protectants andDMSO concentration below 0.1%) and with 50% acetonitrile added toaccount for challenges with solubility of the compound in PBS alone.Stability was determined as the loss of compound with time over a 48hour period with a minimum of 6 time points. The percent remainingcompound at the end of the 48 hours indicated that, with no additives,11.44% of ML291 remained after 48 hours; however, this data wasdependent on the solubility limitations in PBS buffer. With the additionof 50% acetonitrile, to account for solubility, 100% of ML291 remainedafter 48 hours.

Example 10 Synthesis of ML291 Analogs

The high throughput screen identified a first-in-class, potent (760 nMEC₅₀), not generally cytotoxic, submicromolar potent chemical probe,ML291, that selectively activates the apoptotic but not the adaptive armof the UPR, and that moreover demonstrates efficacy in inducing celldeath through activation of the apoptotic in relevant cells. Given thatthe SAR analysis indicated that the nitro moiety on the furan ring wasrequired for its activating activity, and may be the reason for ML291'smodest plasma stability and microsomal instability, analogs of ML291were prepared, which contained the nitro moiety on the furan ring.

ML291 and analogs thereof were generally synthesized by the method shownin Scheme 3. Commercially available 4-substituted piperidines 1 a-d weretreated with an aryl sulfonyl chloride to afford the correspondingsulfonamides which were subsequently reduced to reveal the anilines 2a-d. Conversion to the final products 3 a-f was accomplished by treatinganilines 2 a-f with 5-nitrofuran acyl chloride under microwaveirradiation to generate the final compounds 3 a-f.

N-(4-((4,4-Dimethylpiperidin-1-yl)Sulfonyl)Phenyl)-5-Nitrofuran-2-Carboxamide(SID 134228470; CID 51035286)

4,4-dimethyl-1-((4-nitrophenyl)sulfonyl)piperidine was prepared bycombining 4-nitrobenzenesulfonyl chloride (0.23 g, 1.0 mmol), pyridine(0.12 g, 1.6 mmol) and THF (3 mL). The reaction mixture was stirred atroom temperature while 4,4-dimethylpiperidine (0.15 g, 1.2 mmol) wasadded drop-wise over 10 minutes. The reaction was subsequently heated to60° C. for 20 minutes and monitored by TLC. Upon completion, thereaction cooled to room temperature, was diluted with EtOAc (10 mL) andwashed with saturated NaHCO₃ (10 mL). The EtOAc layer was separated anddried with MgSO₄, filtered, adsorbed to silica and purified by silicagel flash column chromatography (15 minutes, 0-30% v/v EtOAc/hexanes) toproduce pure 4,4-dimethyl-1-((4-nitrophenyl)sulfonyl)piperidine (0.25 g,0.84 mmol, 81% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.39 (d, J=9.0 Hz,2H), 7.96 (d, J=9.0 Hz, 2H), 3.07 (t, J=5.7 Hz, 4H), 1.46 (t, J=5.8 Hz,4H), 0.86 (s, 6H).

4-((4,4-dimethylpiperidin-1-yl)sulfonyl)aniline

The 4,4-dimethyl-1-((4-nitrophenyl)sulfonyl) piperidine (0.12 g, 0.40mmol) was added to a vial and with MeOH:DCM (3 mL:1 mL) and the reactionwas cooled to 0° C. The Raney Nickel (0.002 g, 0.040 mmol) was addedfollowed by portion-wise addition of sodium borohydride (0.030 g, 0.80mmol). The reaction was stirred at 0° C. for 30 minutes and was thendiluted with CH₂Cl₂ (10 mL) and filtered slowly. The CH₂Cl₂ layer waswashed with water (10 mL), dried with MgSO₄, filtered, adsorbed tosilica and purified by silica gel flash column chromatography (15minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pure4-((4,4-dimethylpiperidin-1-yl)sulfonyl)aniline (0.11 g, 0.39 mmol, 98%yield). ¹H NMR (400 MHz, CDCl₃): δ 7.35 (d, J=8.7 Hz, 2H), 6.64 (d,J=8.6 Hz, 2H), 6.03 (s, 2H), 2.80 (t, J=5.6 Hz, 4H), 1.34 (t, J=5.7 Hz,4H), 0.78 (s, 6H).

N-(4-((4,4-dimethylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide

To a microwave vial was added4-((4,4-dimethylpiperidin-1-yl)sulfonyl)aniline (0.11 g, 0.39 mmol),5-nitro-2-furoyl chloride (0.076 g, 0.44 mmol) and acetonitrile (3 mL).The vial was sealed and heated to 150° C. in the microwave for 20minutes. The reaction was cooled to room temperature and was dilutedwith CH₂Cl₂ (10 mL) and washed with saturated NaHCO₃ (10 mL). The CH₂Cl₂layer was separated, dried with MgSO₄, filtered and adsorbed to silicagel. The crude product was purified by silica gel flash columnchromatography (20 minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pureN-(4-((4,4-dimethylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide(0.10 g, 0.25 mmol, 63% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 10.99 (s,1H), 8.02 (d, J=8.9 Hz, 2H), 7.84 (d, J=3.9 Hz, 1H), 7.78 (d, J=8.8 Hz,2H), 7.71 (d, J=3.9 Hz, 1H), 2.91 (t, J=5.5 Hz, 4H), 1.36 (t, J=5.6 Hz,4H), 0.79 (s, 6H). ¹³H NMR (100 MHz, DMSO-d₆: δ 155.0, 151.9, 147.3,141.9, 130.8, 128.5, 120.3, 117.2, 113.4, 42.3, 37.1, 27.8, 27.1. LCMSretention time: 3.307 min. LCMS Purity at 214 nm: 98.3%. HRMS: m/z calcdfor C₁₈H₂₂N₃O₆S (M+H⁺) 408.1224. found 408.124. Melting point: 145-150°C.

N-(4-((4-Fluoropiperidin-1-yl)Sulfonyl)Phenyl)-5-Nitrofuran-2-Carboxamide

4-fluoro-1-((4-nitrophenyl)sulfonyl)piperidine

To a vial was added 4-nitrobenzenesulfonyl chloride 2 (0.13 g, 0.58mmol), pyridine (0.12 g, 1.5 mmol) and THF (2 mL). The reaction wasstirred at room temperature while 4-fluoropiperidine HCl (0.097 g, 0.70mmol) was added over 10 minutes. The reaction was subsequently heated to60° C. for minutes and monitored by TLC. Upon completion, the reactionwas cooled to room temperature, was diluted with EtOAc (10 mL) andwashed with saturated NaHCO₃ (10 mL). The EtOAc layer was separated,dried with MgSO₄, filtered, adsorbed to silica and purified by silicagel flash column chromatography (15 minutes, 0-30% v/v EtOAc/hexanes) toproduce pure 4-fluoro-1-((4-nitrophenyl)sulfonyl)piperidine (0.15 g,0.52 mmol, 89% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.45 (d, J=9.0 Hz,2H), 8.03 (d, J=8.9 Hz, 2H), 4.83-4.68 (m, 1H), 3.15 (m, 2H), 2.97 (m,2H), 1.98-1.78 (m, 4H).

4-((4-fluoropiperidin-1-yl)sulfonyl)aniline

The 4-fluoro-1-((4-nitrophenyl)sulfonyl)piperidine (0.15 g, 0.52 mmol)was added to a vial and with MeOH:CH₂Cl₂ (3 mL:1 mL) and the reactionwas cooled to 0° C. The Raney Nickel (0.003 g, 0.052 mmol) was added,followed by portion-wise addition of sodium borohydride (0.039 g, 1.0mmol). The reaction stirred at 0° C. for 30 minutes and was then dilutedwith CH₂Cl₂ (10 mL) and filtered slowly. The CH₂Cl₂ layer was washedwith water (10 mL), separated, dried with MgSO₄, filtered, adsorbed tosilica and purified by silica gel flash column chromatography (15minutes, 0-5% v/v MeOH/DCM) to produce pure4-((4-fluoropiperidin-1-yl)sulfonyl)aniline (0.13 g, 0.51 mmol, 98%yield). ¹H NMR (400 MHz, CDCl₃): δ 7.36 (d, J=8.7 Hz, 2H), 6.64 (d,J=8.7 Hz, 2H), 6.08 (s, 2H), 4.79-4.64 (m, 1H), 2.94 (m, 2H), 2.80 (m,2H), 1.93-1.72 (m, 4H).

N-(4-((4-fluoropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide

To a microwave vial was added4-((4-fluoropiperidin-1-yl)sulfonyl)aniline (0.039 g, 0.15 mmol),5-nitro-2-furoyl chloride (0.029 g, 0.17 mmol) and acetonitrile (3 mL).The vial was sealed and heated to 150° C. in microwaves for 20 minutes.The reaction was then cooled to room temperature and was diluted withCH₂Cl₂ (10 mL) and washed with saturated NaHCO₃ (10 mL). The CH₂Cl₂layer was separated, dried with MgSO₄, filtered and adsorbed to silicagel. The crude product was purified by silica gel flash columnchromatography (20 minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pureN-(4-((4-fluoropiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide(0.030 g, 0.076 mmol, 49% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 11.02 (s,1H), 8.04 (d, J=8.9 Hz, 2H), 7.84 (d, J=3.9 Hz, 1H), 7.80 (d, J=8.8 Hz,2H), 7.73 (d, J=4.0 Hz, 1H), 4.82-4.68 (m, 1H), 3.07 (m, 2H), 2.94 (m,2H), 1.98-1.78 (m, 4H). ¹³H NMR (100 MHz, DMSO-d₆: δ 155.1, 152.0,147.4, 142.4, 130.2, 128.7, 120.5, 117.3, 113.4, 86.8 (d, J=163.5 Hz),42.2 (d, J=12.6), 30.1 (d, J=32.4 Hz). LCMS retention time: 3.001 min.LCMS Purity at 214 nm: 96.7%. HRMS: m/z calcd for C₁₆H₁₇N₃O₆S (M+H⁺)398.0817. found 398.0845. Melting point: 228-232° C.

N-(4-((4-tert-butylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide

4-tert-butyl-1-((4-nitrophenyl)sulfonyl)piperidine

To a vial was added 4-nitrobenzenesulfonyl chloride 2 (0.20 g, 0.91mmol), pyridine (0.22 g, 2.7 mmol) and THF (4 mL). The reaction wasstirred at room temperature while 4-tert-butylpiperidine HCl (0.16 g,0.91 mmol) was added over 10 minutes. The reaction was subsequentlyheated to 60° C. for minutes and monitored by TLC. Upon completion, thereaction cooled to room temperature, was diluted with EtOAc (10 mL) andwashed with saturated NaHCO₃ (10 mL). The EtOAc layer was separated,dried with MgSO₄, filtered and adsorbed to silica and purified by silicagel flash column chromatography (15 minutes, 0-30% v/v EtOAc/hexanes) toproduce pure 4-tert-butyl-1-((4-nitrophenyl)sulfonyl)piperidine (0.088g, 0.27 mmol, 30% yield). ¹H NMR (400 MHz, CDCl₃): δ 8.37 (d, J=8.9 Hz,2H), 7.94 (d, J=8.9 Hz, 2H), 3.93-3.89 (m, 2H), 2.22 (td, J₁=12.2 Hz,J₂=2.4 Hz, 2H), 1.76-1.72 (m, 2H), 1.38 (qd, J₁=12.4 Hz, J₂=4.1 Hz, 2H),0.89 (m, 1H), 0.82 (s, 9H).

4-((4-tert-butylpiperidin-1-yl)sulfonyl)aniline

The 4-tert-butyl-1-((4-nitrophenyl)sulfonyl)piperidine (0.088 g, 0.27mmol) was added to a vial and with MeOH:CH₂Cl₂ (3 mL:1 mL) and thereaction was cooled to 0° C. The Raney Nickel (0.002 g, 0.027 mmol) wasadded followed by portionwise addition of sodium borohydride (0.020 g,0.54 mmol). The reaction stirred at 0° C. for 30 minutes and was thendiluted with CH₂Cl₂ (10 mL) and filtered slowly. The CH₂Cl₂ layer waswashed with water (10 mL), separated, dried with MgSO₄, filtered,adsorbed to silica and purified by silica gel flash columnchromatography (15 minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pure4-((4-tert-butylpiperidin-1-yl)sulfonyl)aniline (0.068 g, 0.23 mmol, 85%yield). ¹H NMR (400 MHz, CDCl₃): δ 7.52 (d, J=8.7 Hz, 2H), 6.68 (d,J=8.7 Hz, 2H), 4.13 (s, 2H), 3.81-3.77 (m, 2H), 2.12 (td, J₁=12.2 Hz,J₂=2.4, 2H), 1.71-1.67 (m, 2H), 1.36 (qd, J₁=12.4, J₂=4.0 Hz, 4H), 0.87(tt, 31=12.2 Hz, J₂=3.3 Hz, 1H), 0.81 (s, 9H).

N-(4-((4-tert-butylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide

To a microwave vial was added4-((4-tert-butylpiperidin-1-yl)sulfonyl)aniline (0.068 g, 0.23 mmol),5-nitro-2-furoyl chloride (0.037 g, 0.21 mmol) and acetonitrile (2 mL).The vial was sealed and heated to 150° C. in microwaves for 20 minutes.The reaction was then cooled to room temperature, diluted with CH₂Cl₂(10 mL) and washed with saturated NaHCO₃ (10 mL). The CH₂Cl₂ layer wascollected, dried with MgSO₄, filtered and adsorbed to silica gel. Thecrude product was purified by silica gel flash column chromatography (20minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pureN-(4-((4-tert-butylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide(0.060 g, 0.138 mmol, 66% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 10.98 (s,1H), 8.01 (d, J=8.9 Hz, 2H), 7.83 (d, J=4.0 Hz, 1H), 7.76 (d, J=8.8 Hz,2H), 7.70 (d, J=4.0 Hz, 1H), 3.71 (d, J=11.6, 1H), 2.12 (t, J=11.8 Hz,2H), 1.67 (d, J=12.2 Hz, 2H), 1.19 (qd, J, =12.5 Hz, J₂=3.6 Hz, 2H),0.91 (t, J=12.2 Hz, 1H), 0.78 (s, 9H). ¹³H NMR (125 MHz, DMSO-d₆): δ155.0, 152.0, 147.3, 142.0, 130.5, 128.7, 120.3, 117.3, 113.4, 54.9,46.8, 44.8, 31.8, 27.0, 25.7. LCMS retention time: 3.517 min. LCMSPurity at 214 nm: 97.8%. HRMS: m/z calcd for C₂₀H₂₆N₃O₆S (M+H⁺)436.1537. found 436.1533. Melting point: 236-239° C.

N-(4-((piperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide (SID134228467; CID 9415121)

To a microwave vial was added 4-((piperidin-1-yl)sulfonyl)aniline (0.13g, 0.53 mmol), 5-nitro-2-furoyl chloride (0.10 g, 0.58 mmol) andacetonitrile (2 mL). The vial was sealed and heated to 150° C. in themicrowave for 20 minutes. The reaction was cooled to room temperatureand was diluted with CH₂Cl₂ (10 mL) and washed with saturated NaHCO₃ (10mL). The CH₂Cl₂ layer was separated, dried with MgSO₄, filtered andadsorbed to silica gel. The crude product was purified by silica gelflash column chromatography (20 minutes, 0-5% v/v MeOH/CH₂Cl₂) toproduce pureN-(4-((piperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide (0.17g, 0.44 mmol, 82% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 10.99 (s, 1H),8.01 (d, J=8.8 Hz, 2H), 7.84 (d, J=3.9 Hz, 1H), 7.76 (d, J=8.8 Hz, 2H),7.71 (d, J=4.0 Hz, 1H), 2.88 (t, J=5.3, 4H), 1.55-1.53 (m, 4H),1.37-1.35 (m, 2H). ¹³H NMR (125 MHz, DMSO-d₆): δ 155.0, 152.0, 147.3,142.0, 130.5, 128.6, 120.4, 117.3, 113.4, 46.6, 24.7, 22.9. LCMSretention time: 3.131 min. LCMS Purity at 214 nm: 94.1%. HRMS: m/z calcdfor C₁₆H₁₈N₃O₆S (M+H⁺) 380.0911. found 390.0940. Melting point: 183-187°C.

N-(4-((4-methylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide(SID 134228466; CID 17146663)

To a microwave vial was added4-((4-methylpiperidin-1-yl)sulfonyl)aniline (0.11 g, 0.43 mmol),5-nitro-2-furoyl chloride (0.082 g, 0.47 mmol) and acetonitrile (2 mL).The vial was sealed and heated to 150° C. in microwaves for 20 minutes.The reaction was then cooled to room temperature, diluted with CH₂Cl₂(10 mL) and washed with saturated NaHCO₃ (10 mL). The CH₂Cl₂ layer wasseparated, dried with MgSO₄, filtered and adsorbed to silica gel. Thecrude product was purified by silica gel flash column chromatography (20minutes, 0-5% v/v MeOH/CH₂Cl₂) to produce pure N—(4-((4-methylpiperidin-1-yl)sulfonyl)phenyl)-5-nitrofuran-2-carboxamide(0.14 g, 0.37 mmol, 86% yield). ¹H NMR (400 MHz, DMSO-d₆): δ 10.99 (s,1H), 8.01 (d, J=8.9 Hz, 2H), 7.83 (d, J=4.0 Hz, 1H), 7.76 (d, J=8.8 Hz,2H), 7.71 (d, J=3.9 Hz, 1H), 3.59 (d, J=11.6 Hz, 2H), 2.19 (td, J,=11.8, J₂=2.2 Hz, 4H), 1.64 (d, J=10.8 Hz, 2H), 1.31-1.27 (m, 1H), 1.13(qd, J₁=12.5 Hz, 3.6 Hz, 2H), 0.85 (d, J=6.4 Hz, 3H). ¹³H NMR (125 MHz,DMSO-d₆): δ 155.0, 152.0, 147.3, 142.0, 130.6, 128.6, 120.4, 117.3,113.4, 46.1, 32.8, 29.3, 21.3. LCMS retention time: 3.184 min. LCMSPurity at 214 nm: 96.6%. HRMS: m/z calcd for C₁₇H₂₀N₃O₆S (M+H⁺)394.1067. found 394.1082. Melting point: 189-193° C.

Example 11 Activity of ML291 Analogs

Five analogues of probe ML291 were prepared. All five compounds,including the probe, were synthesized and their associated data, alongwith the probe, are summarized in Table 9.

TABLE 9 EC50 (μM) Compound UPR CHOP Assay^(a) UPR XBP1 Assay^(a) Amt(mg) ML291 0.78 >80 21.1 Analog 1 0.76 >80 22.2 Analog 2 0.74 >80 23.2Analog 3 0.43 >80 21.9 Analog 4 0.65 >80 22.5 Analog 5 0.77 >80 22.5Data is an average of four runs (n = 4) for the UPR CHOP and UPR XBPassays. ^(a)Conditions: F12 nutrient mix HAMs supplemented with 10%hi-FBS, 1X penicillin/streptomycin, 1X MEM-NEAA.

Example 12 Additional ML291 Analogs

Additional analogs of ML291 are presented in Table 10.

TABLE 10 Compound PubChem SID Structure Analog 6 104222717

Analog 7 104222719

Analog 8 104222721

Analog 9 104222726

Analog 10 104222727

Analog 11 104222728

Analog 12 104222729

Analog 13 104222730

Analog 14 104222731

Analog 15 104222733

Analog 16 104222734

Analog 17 117695369

Analog 18 117695370

Analog 19 117695371

Analog 20 117695372

Analog 21 117695373

Analog 22 117695374

Analog 23 117695375

Analog 24 —

Analog 25 —

Analog 26 —

Analog 27 —

Analog 28 —

Analog 29 —

Analog 30 —

To further enhance potency and efficacy, lead optimization studies arecarried out, wherein common nitro group isosteres such as nitrile andcarboxylic acid groups, along with pyridine derivatives are prepared,which have been reported as successful surrogates for aryl nitrofunctionality (Meanwell (2011) J. Med. Chem. 54:2529-2591).Additionally, the nitro could be replaced with ester or sulfonamide,sulfone, an approach that was very successful in the development of theBCL2 inhibitor ABT-263 (Oltersdorf, et al. (2005) Nature 435:677-681).

What is claimed is:
 1. A selective activator of the apoptotic arm of theUnfolded Protein Response, wherein said activator has the structure ofFormula I, or a pharmaceutically acceptable salt, ester or prodrugthereof,

wherein X is C; R¹ is a hydrogen; and R² is a substituted alkyl group.2. A pharmaceutical composition comprising a pharmaceutically acceptablecarrier and a compound of Formula I, or a pharmaceutically acceptablesalt, ester or prodrug thereof,

wherein X is C, N or O; and R¹ and R² are each independently a hydrogen,hydroxyl, halo, or a substituted or unsubstituted alkyl or alkoxy group;or R¹ and R² together form a substituted or unsubstituted cycloalkylgroup, with the proviso that when X is O, R¹ and R² are absent.
 3. Thepharmaceutical composition of claim 2, wherein said composition isformulated for intratumoral, intracerebral, intracerebroventricular, orintrathecal administration.
 4. A method for synthesizing a compound ofFormula I comprising: (a) treating a 4-substituted piperidine with anaryl sulfonyl chloride to produce a sulfonamide; (b) contacting thesulfonamide with a reducing agent to produce an aniline; and (c)treating the aniline with 5-nitrofuran acyl chloride under microwaveirradiation, thereby producing a compound of Formula I

wherein X is C; and R¹ and R² are each independently a hydrogen,hydroxyl, halo, or a substituted or unsubstituted alkyl or alkoxy group;or R¹ and R² together form a substituted or unsubstituted cycloalkylgroup.
 5. A method for selectively activating the apoptotic, but not theadaptive arm, of the Unfolded Protein Response (UPR) comprisingcontacting a cell with a compound of Formula I thereby selectivelyactivating the apoptotic, but not the adaptive arm, of the UPR,

wherein X is C, N or O; and R¹ and R² are each independently a hydrogen,hydroxyl, halo, or a substituted or unsubstituted alkyl or alkoxy group;or R¹ and R² together form a substituted or unsubstituted cycloalkylgroup, with the proviso that when X is O, R¹ and R² are absent.
 6. Amethod of treating or lessening the severity of a Unfolded ProteinResponse-related disease in a subject comprising administering to asubject in need of treatment a pharmaceutical composition of claim 2thereby treating or lessening the severity of the subject's unfoldedprotein response-related disease.
 7. The method of claim 6, wherein theUnfolded Protein Response-related disease is a pre-cancerous syndrome,Alzheimer's disease, stroke, Type 1 diabetes, Parkinson disease,Huntington's disease, amyotrophic lateral sclerosis, myocardialinfarction, cardiovascular disease, atherosclerosis, arrhythmias,hemophilia, age-related macular degeneration or a lysosomal storagedisorder.
 8. The method of claim 6, wherein the Unfolded ProteinResponse-related disease is cancer.
 9. The method of claim 8, furthercomprising co-administering at least one anti-neoplastic agent.